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Functional genomics of cereals-pathogens interactions

Team CEREPATH / Marie Dufresne

Small-grain cereals are economically important crops. Among diseases affecting cereals, Fusarium head blight (FHB) is a major concern: it reduces yields but also causes quality losses due to the production of mycotoxins, including deoxynivalenol (DON), harmful to humans and animals. There is no specific resistance to F. graminearum but a number of quantitative trait loci have been identified, some of which associated with mycotoxins detoxification.

Our research in the past few years aimed at identifying plant genes involved in the detoxification processes of DON and at characterizing their potential involvement in quantitative resistance to FHB. To this end, two cereal species were used: Brachypodium distachyon as a model species for small-grain cereals and bread wheat. Both were used in interaction with the main FHB causal agent in Northern countries, the ascomycete fungus Fusarium graminearum. Most recently, we also initiated scientific projects on tripartite interactions : plant-beneficial microorganism-fungal pathogen with both fundamental and applied research aspects. 

 

 

Cereal UGTs, DON detoxification and resistance to FHB

People involved: J.C. Pasquet, M. Gatti, C. Macadré, J.M. Seng, M. Dufresne

Fundings: PhD fellowship DIM Astrea, Grants4Target Bayer CropScience

 

Through phylogenetics, we have identified B. distachyon genes orthologous to a barley UGT gene and shown that one of them (Bradi5g03300)confers DON resistance when expressed in a yeast system (Schweiger et al. 2013). We then performed functional analyses in B. distachyon. Mutant lines were obtained by screening the TILLING mutant collection (INRA, Versailles and URGV) and overexpressing lines were constructed. This study showed a direct relationship between the level of DON glucosylation and resistance to FHB(Pasquet et al., 2016).


The Bradi5g03300 UGT gene is involved in the control of spike colonization by F. graminearum. A, Typical FHB symptoms on the different lines 7 days after point inoculation by F. graminearum: mock, negative control; Bd21-3, wild-type; 8637-12 and 6829-7, TILLING mutant lines; OE-9R5, 24R27, 10R14, transgenic lines overexpressing the Bradi5g03300 UGT gene. Bars: 1 cm. B, Quantification of FHB symptoms using a scoring scale 7 (gray bars) and 14 (back bars) days after point inoculation (n>30, error bars represent the standard error, different letters indicate significant differences between conditions; Newman and Keuls test, p-value ≤ 0.001).

More precisely, early and efficient conjugation of DON into its glucoside D3G allows the establishment of a strong resistance in B. distachyon, not only to spike colonization but to initial fungal infection. In contrast, stable expression of the Bradi5g03300 gene in wheat only conferred resistance to spike colonization, pointing out plant species-specific mechanisms (Gatti et al., 2018a). Once the processes of detoxification and resistance are better understood in this model plant, results will be more easily transferred to cereal crops, in particular bread wheat. In the frame of M. Gatti’s PhD project (2014-2017), a synteny approach was conducted in collaboration with GDEC unit (Clermont-Ferrand, France). The potential wheat ortholog of the B. distachyon Bradi5g03300 gene was identified and genetically transformed in a B. distachyon ecotype susceptible to FHB. Functional characterization of the transgenic lines showed that the wheat UGT gene conferred resistance to F. graminearum andallowed strong DON reduction in planta (Gatti et al. 2018b, in revision in Frontiers in Plant Science).

 

 

Plant genes as interesting candidates involved in FHB resistance

People involved : V. Changenet, M. Januario, C. Macadré, M. Dufresne

Fundings: PhD fellowship MESRI

 

Transcriptomics analyses were performed on B. distachyon plants either infected with DON-producing or DON non producing strains of F. graminearum or inoculated with DON itselfin order to identify candidate genes potentially involved in detoxification as well as metabolic pathways deregulated by the mycotoxin. A number of differentially expressed genes were retrieved, some of them encoding protein functions known to be involved in detoxification processes such as UGTs, CYPs (cytochrome P450) and MATEs(multidrug and toxic compound extrusion) (Pasquet et al. 2014). Among these candidates, we favoured a gene encoding a CYP (CYP711A29). Such enzymes are usually occurring in the early steps of detoxification processes in plants, allowing a chemical modification of metabolites which are then more amenable to conjugation performed either by UGTs or GSTs. DON exhibiting a number of hydroxyl groups can readily be conjugated. Our hypothesis was that CYP711A29 is able to modify the DON glucoside into an even less toxic molecule. This hypothesis has been tested in the frame of V. Changenet’s PhD work (2015-2018) following a similar strategy as the one used to study Bradi5g03300 (construction of B. distachyon lines over-expressing the gene; search for mutant lines in the TILLING collection; functional characterization of these lines and in vitro characterization of CYP711A29 activity and determination of the end product by LC-MS-MS (collaboration with D. Werck, IBMP, Strasbourg). These analyses have shown that if the B. distachyon CYP711A29 does not detoxify DON nor its glucoside, but is involved in orobanchol biosynthesis (Changenet et al., in preparation). Experiments are underway to determine whether strigolactones are part of the plant defense reactions or if the fungal pathogen diverts this biosynthetic pathway to favour its establishment in the plant tissues.

 

Impact of beneficial microorganisms on the resistance to fungal pathogens in cereals

People involved : T. Plainchamp, J.M. Seng, M. Dufresne

Fundings: WheatBiocontrol (SATT), Stress’N’Sym (Plant2Pro, Carnot)

 

More recently, our research team has focused on the imapct of beneficial micro-organisms on the interactions between cereals and fungal pathogens. A first aspect, corresponding to a collaborative project with B Lefèbvre (LIPM, Toulouse, France) and T Girin-S Ferrario-Méry (IJPB? Versailles, France), aims at analyzing the genetics of B. distachyon response to mycorhization as well as the impact of endomycorhizal fungi on the resistance of the model cereal species to fungal pathogens.


Fusarium Head Blight symptoms observed on wheat spikes 14 days after spray inoculation with Fusarium graminearum with (right) or without prior treatment of the spikes by the biocontrol agent. Bars: 1 cm.

A second projet with both fundamental and applied research aspects, studies the efficiency of an innovative biocontrol solution against FHB on wheat and analyzes the possible mode of action on the wheat-F. graminearum interaction.